Research ArticleANTIMICROBIALS

Identification of biologic agents to neutralize the bicomponent leukocidins of Staphylococcus aureus

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Science Translational Medicine  16 Jan 2019:
Vol. 11, Issue 475, eaat0882
DOI: 10.1126/scitranslmed.aat0882
  • Fig. 1 Structure and sequence alignments of selected potent anti-leukocidin centyrins.

    (A) Crystal structure of parental TENCON (Protein Data Bank: 3TES). Purple regions indicate sites of amino acid diversification introduced into panning libraries used to identify centyrins with strong affinities for leukocidins. (B) Amino acid sequence alignment of selected leukocidin centyrins and the parental TENCON sequence. Positions of loop regions are underlined and labeled. Amino acid residues conserved across all sequences are highlighted in yellow. Numbers in parentheses indicate the position of the amino acid in the centyrins.

  • Fig. 2 Ability of centyrins to block leukocidin-mediated lysis of primary hPMNs.

    (A to E) Viability of primary hPMNs after a 1-hour incubation with indicated doses of LukAB (A), LukED (B), HlgAB (C), HlgCB (D), or PVL (E) and in the presence of increasing concentrations of centyrins. Data were normalized to the maximal lactate dehydrogenase release by each individual toxin. (F) EC50 of the selected most potent neutralizing centyrins calculated from nonlinear robust regression fit of the dose-response curve (A to E). (G) EC50 of identified LukED/HIgAb cross-reactive centyrins calculated from nonlinear robust regression fit of a dose-response curve (B and C). (H) EC50 of identified cross-reactive centyrins between PVL/HlgCB calculated from nonlinear robust regression fit of dose-response curve (D and E). (A to H) Results are the means ± SEM, with n = 4 donors.

  • Fig. 3 Ability of centyrins to block LukED- and HlgAB-mediated hemolysis.

    Primary human erythrocytes were incubated with 90% lytic dose (LD90) of LukED (A) or HlgAB (B) in the presence of the indicated centyrin, and hemolysis was evaluated after a 30-min incubation at 37°C by measuring the absorbance of released hemoglobin into the surrounding media at 450 nm. Hemolysis was normalized to Triton X-100 lysed control. Results are the means ± SEM, with n = 6 different donors. EC50 of the most potent neutralizing centyrins are shown to the right.

  • Fig. 4 Mechanism of neutralization by anti-leukocidin centyrins.

    (A) Binding of LukAB to the human CD11b I-domain in the presence of TENCON or LukAB centyrin (SM1S17). Results are from two independent experiments. (B) Immunoblot of biotinylated toxin (anti-his: 100-ng protein loaded; streptavidin: 50-ng loaded; white line indicates noncontinuous lanes) ladder-like appearance of the toxins is the product of varying degrees of biotinylation. (C) Viability of hPMNs after 1-hour incubation with unlabeled and biotinylated S-subunits (Xb) paired with their cognate F-subunit. Results are the means ± SEM, with n = 4 donors. (D to G) Indicated biotinylated subunits were incubated with hPMNs in the presence of TENCON, the neutralizing centyrin or unlabeled toxin subunit (competitor), and binding of the labeled toxin to the cell surface evaluated via flow cytometry. Results are presented as the median fluorescence intensity ± SEM [(D) n = 5 donors, LukE (unlabeled) n = 2 donors; (E) n = 5 donors, HlgA (unlabeled) n = 2 donors; (F and G) n = 4 donors].

  • Fig. 5 Ability of anti-leukocidin centyrins to protect hPMNs from S. aureus–mediated killing.

    (A) Extracellular infection of hPMNs with indicated USA300 strains at a multiplicity of infection (MOI) of 25. Bars indicate means ± SEM, with n = 9 donors. (B) Extracellular infection of hPMNs with wild-type USA300 at MOI of 25 in the presence of indicated centyrins. Bars indicate means ± SEM, with n = 9 donors. ns, not significant; WT, wild-type. (C) Extracellular infection of hPMNs with wild-type USA300 at MOI of 25 in the presence of titrated concentrations of TENCON and SM1S17. Points indicate means ± SEM, with n = 3 donors. (D) Extracellular infection of hPMNs with MRSA and MSSA strains of diverse clonal complexes at MOI of 25 with TENCON or SM1S17. Bars indicate means ± SEM, with n = 6. Statistical analysis probing for neutralizing effect of SM1S17 was done using a one-tailed paired t test. (E) Infection of hPMNs with indicated USA300 isogenic strains pre-opsonized with 20% human serum (MOI of 15) in the presence of indicated centyrins, without or with subsequent centrifugation. Bars indicate means ± SEM, with n = 4 donors. (A to E) Cell viability was determined with CytoTox-ONE homogeneous membrane integrity assay. Statistical analysis where applicable was examined using ordinary one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Protection from in vivo intoxication with ABDcon-centyrins.

    (A) Survival curves of mice after intravenous administration of centyrins at 10× (30 μg) or 1× (3 μg) premixed with a lethal dose of purified LukED (10 μg per subunit); n = 3 mice per group. PBS, phosphate-buffered saline. (B) Survival curves of mice treated with a prophylactic dose of LukE centyrins (~500 μg or ~100 μg) before intravenous administration of a lethal dose of LukED (10 μg per subunit); n = 3 mice per group. (C) Viability of hPMNs following incubation with purified LukED (76 nM, 2.5 μg/ml) and SM1S26 with or without ABDcon. Results indicate means ± SEM; n = 3 donors. (D) Survival curves of mice that had been treated prophylactically with ABDcon-SM1S26 (at molar excesses of 1×, 10×, and 100×) 5 hours before multiple sequential intravenous administrations (indicated by arrows) of purified LukED (6 μg per subunit); n = 3 mice per group. (E and F) Survival curves of mice treated prophylactically with ABDcon-centyrins (at molar excess of 50×) 48 hours before first intravenous administration of lethal dose of purified toxin (6 μg per subunit), LukED (E), or HlgAB (F) with cross-reactive centyrins color matched across panels; surviving mice were administered additional lethal doses of toxin at 24-hour intervals and monitored up to 2 days after intoxication; n = 4 mice per group.

  • Fig. 7 Protection from in vivo infection with ABDcon-centyrins.

    (A) Survival curves of mice infected intravenously with ~4 × 107 colony-forming units (CFU) of S. aureus strain Newman. Centyrins were administered intravenously 4 hours before and 48, 96, and 144 hours after infection. n = 12 to 15 mice per group. (B) Survival curves of mice infected intravenously with ~3 × 107 CFU of USA500. Centyrins were administered intravenously 4 hours before and 24, 72, and 120 hours after infection. n = 9 to15 mice per group. (C) Survival curves of mice infected intravenously with ~2 × 107 CFU of USA500. Treatments were given intravenously 4 hours before and 24, 72, and 120 hours after infection. n = 10 mice per group. (A to C) Statistics were performed using Gehan-Breslow-Wilcoxon test with P values adjusted for multiple comparisons. (D) Mice were infected intravenously with ~1 × 107 CFU of USA500. Treatments were as in (C). CFU in tissues were measured 96 hours after infection. CFU from individual mice is shown ± SEM. n = 12 to 15 mice per group. LOD, limit of detection. (E) Mice were infected with ~8 × 107 CFU of USA300 premixed with ABDcon-centyrins, ABDcon-TENCON, or a mixture of ABDcon-SM1S26 and SM1S163, intraperitoneally (ip). CFU in tissues were measured 48 hours after infection. CFU from individual mice are shown ± SEM. n = 10 to 11 mice per group. Statistical analysis of bacterial burden was examined using two-tailed Mann-Whitney test, *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Table 1 Summary of centyrin panning campaign.

    Target
    leukocidin
    Panning output*Selective
    neutralizers†
    Cross-reactive
    neutralizers‡
    LukABLukAB binders: 2610
    LukEDLukE binders: 7172 (LukED and
    HlgAB)
    LukD binders: 44
    HlgABHlgA binders: 5130
    HlgB binders: 13
    HlgCB16234 (HlgCB and
    LukSF-PV)
    HlgC binders: 309
    LukSF-PV
    (PVL)
    LukS binders: 2298
    LukF binders: 28

    *Clones of unique binders, from library variants of TENCON, of target leukocidins by in vitro library selection system (CIS display).

    †Purified centyrins found to selectively neutralize cytotoxicity on hPMNs by leukocidins.

    ‡Purified centyrins found to neutralize cytotoxicity on hPMNs by two leukocidin.

    Supplementary Materials

    • The PDF file includes:

      • Methodological Details
      • Fig. S1. Selected neutralizing LukE/D centyrins from loop diversified library.
      • Table S1. Reported centyrin Kds calculated from BLI.
      • Table S2. S. aureus strains used in the study.
      • Legend for table S3
      • References (4554)

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S3. Primary data (provided as an Excel file).

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